CHARACTERIZATION OF DNA POLYMERASE ETA CONTRIBUTION TO GENOME STABILITY UNDER REPLICATION STRESS

pol η is a Y-family translesion DNA polymerase extensively characterized for its ability to bypass thymine dimers induced by UV light. More recently, pol η has also been demonstrated to perform numerous functions independent of DNA lesions. We have shown that in budding yeast pol η sustains genome duplication when dNTP pools are reduced by hydroxyurea, but this activity favors the inclusion/stabilization of RNA stretches in genomic DNA that become toxic if not removed by RNase H enzymes. The origin of such RNA substrates and the exact mechanism by which they trigger genome instability are still unclear. In this study, I found that, upon dNTPs shortage, pol η may act on R-loops to promote resolution of transcription-replication collisions, ultimately causing RNA moieties to remain embedded in DNA behind the replication fork. The techniques developed to study ribonucleotides incorporated in DNA molecules, do not allow to directly identify pol η-dependent RNA stretches embedded in DNA. To develop a strategy to overcome such limitation, we assessed the feasibility of detecting ribonucleotides embedded in DNA exploiting direct sequencing with Oxford Nanopore Technologies (ONT). To this extent, I synthesized DNA molecules containing rNMPs at known positions and we elaborated appropriate data analysis pipelines to recognize ribonucleotides in DNA by ONT. We reported for the first time that ONT can identify all four ribonucleotides incorporated in DNA by capturing rNMPs-related alterations in base calling, current intensities, and dwell times. Finally, I investigated how this novel activity of pol η may be regulated. We have previously observed that budding yeast pol η can be mono-SUMOylated and that SUMOylation levels drop in hydroxyurea. I found that this is independent of the presence/absence of RNases H in cells and that pol η SUMOylation is maintained in different cell cycle phases. I also identified K603 as the main candidate residue for this modification.